Albumin is the most abundant protein in blood plasma and is found at a concentration of approximately 35 to 55 grams per liter of plasma. Albumin normally is a monomer with a molecular weight of 66,000 to 69,000; however, it may also form polymers, with proportionally higher molecular weights. The most common method for purification of albumin from plasma is by the Cohn, cold ethanol precipitation, method (Cohn et al., J. Amer. Chem. Soc., 68, 459-475 (1946); also U.S. Pat. No. 2,710,294). The Cohn method separates plasma proteins by sequential precipitations using increasing concentrations of cold ethanol and decreasing pH values. The fractions separated by this method are: Fraction I, Fraction II+III, Fraction IV.sub.1, Fraction IV.sub.4, and Fraction V. Most of the albumin present in plasma is in the Fraction V precipitate or paste.
The Fraction V albumin extracted from plasma by the Cohn method is estimated to be 95% pure (i.e., 95% by weight of the protein present in Fraction V is albumin). However, further purification of albumin is desirable, since pasteurization of the albumin solutions results in precipitation of a portion of the contaminating proteins, which adversely affects the clarity of the final albumin solutions.
Albumin's main uses are as a plasma extender and for correction of hypoproteinemia. In addition, albumin is frequently used: (1) as a stabilizing agent for other proteins contained in preparations administered for various medical treatments, such as Factor VIII; (2) to maintain the colloid osmotic pressure; and (3) for in vivo transport functions, for example, of fatty acids and drugs.
Methods that have been used to further purify albumin from Fraction V include: additional ethanol precipitations or acetone precipitations with heating. The yield of albumin from the additional ethanol precipitation method is relatively low, however, due to denaturation and loss of albumin during the precipitation procedure.
The acetone precipitation with heating method of purifying albumin uses two steps. The first step is to resuspend the albumin precipitate in acetone, where acetone-soluble contaminants remain in solution. The albumin precipitate is separated from the acetone-soluble contaminants by filtration, and the recovered albumin precipitate is redissolved in water. The second step is the heating step, during which additional impurities are removed. During the heating step, a portion of the remaining impurities precipitate, due to heat denaturation, while albumin, which is relatively stable to heat denaturation, remains in solution. This purification method also results in a low yield of albumin, mainly due to denaturation of a portion of the albumin. In addition, the heating step reduces the albumin monomer content and increases the albumin polymer content. High monomer contents are desired in the final albumin product, since it has been suggested that albumin polymers are cleared more rapidly from circulation than are albumin monomers, effectively resulting in a reduction in the concentration of infused albumin. It has also been suggested that the polymer form of albumin may produce an undesirable immunological response.
Other methods that have been used for the separation of albumin from the impurities in Fraction V have generally been by chromatography, and most commonly, by ion-exchange chromatography. Two general forms of ion-exchange chromatography are used. The first is by the use of cation-exchange resins, which contain a negatively-charged ligand attached to a support matrix, and the other is by use of anion-exchange resins, which contain a positively-charged ligand attached to a support matrix.
To determine if a protein will bind to an ion-exchange resin (either an anion- or a cation-exchange resin), the isoelectric point (pI) of the desired protein, the pH of the solution, and the salt concentration of the solution must be considered.
The pI of a protein is the pH at which there is a net zero charge on the protein. The pI values of proteins, which are specific for a particular protein, vary over the whole spectrum of the pH range. By varying the pH of a solution, the charge on a particular protein can be manipulated and utilized for the purification of a desired protein from contaminating proteins. When the pH of the solution containing the selected protein equals its pI, the net charge on the protein is zero, and the protein will not bind to either an anion- or a cation-exchange column. When the pH of the protein solution is decreased to below the pI of the selected protein, there will be a net positive charge on the protein which increases as the pH decreases. Under these conditions, proteins with a net positive charge will bind to a cation-exchange resin. The strength of binding to the resin is dependent on the total charge on the protein, i.e., if the PH is just below the pI of the protein, there will only be a small positive charge on the protein, and the binding to a cation-exchange resin will be very weak. If the pH of the solution is far below the pI of the protein, there will be a large positive charge on the protein, and the binding, to a cation-exchange resin, will be strong. When there is a net positive charge on the protein, the protein will have no affinity for an anion-exchange resin. Conversely, as the pH of the protein solution is increased above the pI of the protein, by the addition of alkali, there will be a negative charge on the protein which increases as the pH increases. As a result of this negative charge, the protein will be able to bind to an anion-exchange resin. The strength of the binding is dependent on the strength of the charge on the protein. A negatively-charged protein will have no affinity for a cation-exchange resin.
An additional effect of manipulating the pH of the solutions is that some proteins can be induced to precipitate. Many proteins are insoluble at pH values that approach their pI value. Many proteins will, therefore, precipitate from solution when the pH of the solution is adjusted to a value equal to their pI. Albumin remains soluble when pH values approach its pI.
Proteins, even when charged, can be prevented from binding to an ion-exchange resin by the presence of salt in the solution. In ion-exchange chromatography, salt competes with the charged protein for binding to the charged groups of the chromatography resin. When a protein has a small charge, the binding to the ion-exchange resin will be weak, and only low salt concentrations will be required to compete with the protein to prevent binding to the ion-exchange resin. Conversely, when a protein has a larger charge, the binding to the ion-exchange resin will be strong, and high salt concentrations will be required to compete with the protein to prevent binding to the ion-exchange resin.
In view of the foregoing, it can be seen that pH values can be chosen that will result in a protein having either a negative, positive, or zero charge, or that will make the protein insoluble, and salt concentrations can be chosen that will permit binding of a charged protein to an ion-exchange resin.
Although ion-exchange chromatography has previously been used to purify albumin, the known methods are undesirably expensive and inefficient. For example, in one method, the albumin (instead of contaminating proteins) is bound to the ion-exchange resin. This method requires the use of large amounts of resin to bind the large amounts of albumin present in the impure albumin solution. This results in undesirably-high resin costs and long reaction times.
A more efficient and economical method of purifying albumin by ion-exchange chromatography, is to bind the contaminants, which are present in the impure albumin solutions in much smaller amounts than albumin, to the resin. In prior-art processes in which contaminants are bound, both anion- and cation-exchange resins are used in combination. When these prior-art anion/cation procedures were used, salt, at concentrations up to 70 mm, was added to the albumin solutions. This method also results in an undesirably-high cost, since the added salt (1) must be removed prior to medical use of the albumin; and (2) prevents binding of some contaminants to the resin, resulting in undesirably-low purities of the final albumin solution.
None of the prior-art methods described above result in albumin solutions where greater than 99% of the protein present is albumin or where the albumin monomer content is desirably high. In addition, some contaminants, such as alpha-1-acid glycoprotein, have not been successfully removed from prior-art albumin concentrates. The presence of alpha-1-acid glycoprotein in the final albumin product is undesirable, since it has a high affinity for many basic drugs and, when infused with albumin, results in unpredictable circulating concentrations of such drugs.
Currently, the methods used to purify albumin result in final products having either undesirably-high proportions of albumin polymers, and/or undesirably-high levels of particulate matter, and/or an undesirably-low albumin purity. It is therefore desirable that there be provided a process for the separation of albumin monomer from other contaminating proteins found in impure albumin fractions, such as the Fraction V paste produced by the Cohn method.